Device for microbiological analysis

ABSTRACT

The device for the microbiological analysis of a support ( 41 ) adapted to contain microorganisms marked by a fluorophore agent comprises illuminating means adapted to illuminate said support ( 41 ) comprising two bases ( 21 ) on each of which is mounted at least one group of light sources ( 22 ), with said sources ( 22 ) of that group being regularly spaced from each other to form an array for illuminating said support ( 41 ), said bases ( 21 ) being fixed to a frame of said device, one on each side of an opening ( 9 ) formed in said frame, with said bases ( 21 ) being inclined towards each other in the direction of a predetermined location for reception of said support ( 41 ) in said device.

The present invention concerns a device for the microbiological analysisof supports that may contain microorganisms in order to detect thepresence or absence of those microorganisms.

One way to analyze such supports consists of detecting the presence ofthe microorganisms by analysis of the fluorescence emitted by thosemicroorganisms after they have been marked by what are referred to asfluorogen or fluorophore markers.

These markers have the particularity of fluorescing only when they havebeen activated beforehand by an enzyme contained in the microorganisms.

These markers generally comprise a fluorophore group as well as a groupcapable of concealing or preventing the fluorescence of the fluorophoregroup from showing. When the microorganisms are present, the effect ofthe enzyme thereof is to modify that second group in order that thefluorescence of the first group may be detected.

Thus, as illustrated by the spectra 53 and 54 of FIG. 7, when themicroorganisms so marked are subjected to an appropriate excitationlight, the fluorophore group is capable of absorbing light energy withan absorption spectrum 53 of which the crest 53′ is at a wavelength λ₂and of releasing that energy in the form of a characteristic fluorescentemission spectrum 54 of which the crest 54′ is at a wavelength λ₃,distinct from λ₂.

In order to observe the fluorescence emitted by the microorganisms inpresence of such markers, devices are already known for microbiologicalanalysis (integrated into microscopes, such as in the United Statesapplication US 2007/0153372), comprising illuminating means adapted toilluminate the support to analyze for detecting, by fluorescence, thepresence of microorganisms marked by a fluorophore agent as well as awindow for viewing the light coming from the support in response to thelight emitted by the illuminating means.

The invention concerns the provision of a device for detectingfluorescence, as for the device of the prior art, while having betterperformance and being more practical.

To that end, it provides a device for the microbiological analysis of asupport adapted to contain microorganisms marked by a fluorophore agentcomprising illuminating means adapted to illuminate said support todetect, by fluorescence, the presence of said marked microorganisms aswell as a window for viewing the light emitted by said support inresponse to the excitation light produced by said illuminating means;characterized in that said illuminating means comprise two bases on eachof which is mounted at least one group of light sources, with saidsources of that group being regularly spaced from each other to form anarray for illuminating said support, said bases being fixed to a frameof said device, one on each side of an opening formed in said frame toallow the light emitted by said support to pass through said viewingwindow, with said bases being inclined towards each other in thedirection of a predetermined location for reception of said support insaid device.

The two bases provided with light sources of the device according to theinvention, by being inclined relative to each other and directed towardsthe support to analyze (when it is placed in the device), make itpossible to obtain symmetrical and homogenous illumination of the wholeof the surface of the support to illuminate since the excitation lightproduced by the light sources is emitted from both sides of the openingwhich is provided to enable the observation of the light emitted inresponse to that light excitation.

The fact that those two bases are each provided with a group of lightsources arranged in a regular array also contributes to making theillumination of the whole of the surface of that support particularlyhomogenous.

Such an arrangement thus makes it possible to illuminate supports oflarger size than for the devices of the prior art integrated intomicroscopes while maintaining uniform illumination with a contrast suchthat it is easily possible to detect the presence of microorganisms withthe naked eye or with any other image sensor.

It is in particular made possible to illuminate for example microporousmembranes while keeping an optimum and central location of the openingenabling the observation of the light emitted in response to the lightexcitation produced by the light sources.

According to features that are preferred for reasons of simplicity andconvenience for both manufacture and use:

-   -   said light sources each have a same light spectrum centered on a        predetermined wavelength; and/or    -   said light sources are light emitting diodes.

According to other preferred features, each said base is inclined by anangle between 40° and 50° relative to said predetermined location forreception of said support.

It proves to be the case, surprisingly, that the range of values[40°-50°] for the inclination of the plates relative to the support gavean optimum performance regarding the comfort of reading for thedetection of the microorganisms.

More particularly, it appears that within this range of values veileffect phenomena (from diffusion of extraneous light through the viewingwindow) and reflection phenomena (from the reflection of light on thesupport to analyze) are significantly minimized.

In other words, it is within this range of values that the best imagerendering is obtained, in terms of contrast, homogeneity and brightness.

According to still other preferred features, the height between thecenter of each array and said predetermined location for reception ofsaid support in said device is a function of said support.

According to still other preferred features, the center of each saidarray is situated at a height between 39.75 mm and 69.75 mm from saidpredetermined location for reception of said support.

It has proved to be the case that, given in particular the dimensionsthat are desirable in practice for the support to analyze, this range ofheight provided a good compromise enabling at the same time to obtain acompact device while keeping good performance with regard to contrastand comfort for reading for the easy detection of the microorganisms.

According to still other preferred features:

-   -   on each base there is also mounted, in addition to said group of        light sources, designated first group, another group of light        sources, designated second group, with said sources of said        second group also being regularly spaced from each other to form        a second array for illuminating said support.    -   for each base, the centers of said first and second arrays        coincide;    -   said first and second arrays are interlaced within each other;    -   said illuminating means comprise, for each base, a diffuser for        the light emitted by said sources.    -   each diffuser is a plate of glass on which one of the faces is        sandblasted;    -   said viewing window comprises a filter adapted allow the longest        wavelengths to pass;    -   said viewing window comprises a plurality of filters;    -   said filters belong to a slide slidingly mounted on said frame;    -   said illuminating means are adapted to illuminate the whole of        said predetermined location for reception of said support;        and/or    -   said support to analyze is a microporous membrane.

The features and advantages of the invention will appear from thefollowing description, given by way of preferred but non-limitingexample, with reference to the accompanying drawings in which:

FIG. 1 is a diagram of a device according to the invention;

FIG. 2 is a perspective view of that device beside which is representeda filter unit to analyze in the device;

FIGS. 3 and 4 are respectively a perspective view taken from below and asection view in elevation taken on a median plane of symmetry of thatdevice;

FIG. 5 is a perspective view of one of the two printed circuit boards ofthat device on which are fixed two groups of light emitting diodes, eachemitting light at a predetermined wavelength;

FIG. 6 is a perspective view of another embodiment of the deviceaccording to the invention; and

FIG. 7 illustrates the spectral diagrams of different optical members ofa device according to the invention as well as the spectral diagram ofthe fluorophore agent used with that device to mark the microorganisms,with a common scale of wavelengths along the x-axis and a common scaleof relative light intensity along the y-axis.

A description will now be given of a preferred embodiment of the deviceaccording to the invention with the help of FIGS. 1 to 5, beforedetailing its mode of operation with the help of the spectral diagramsillustrated in FIG. 7.

The device 1 illustrated in FIGS. 1 to 5 comprises a casing 2,illuminating means 3 formed from two illuminating members 4, a slidingdrawer 5 and a viewing window 7.

The casing is of parallelepiped general shape and has an upper wall 10and four side walls 11 to 14, a portion of wall 14 being deliberatelynot shown in order to show the interior of the device.

The viewing window 7 is here constituted by a low-pass filter 6 (that isto say that it allows the lowest frequencies to pass, and thus thelongest wavelengths), an opening 9 being formed in the upper wall 10 ofthe casing 2 to receive that filter 6 therein.

As will be seen below, the interior of this casing 2, delimited by thewalls 10 to 14 and 30, forms an analysis chamber isolated from thesurrounding light and in which the support to analyze is received.

The support to analyze is here a microporous membrane 41 belonging to afilter unit 40, here a unit commercialized by Millipore® under thetrademark Milliflex®. This membrane 41 has a diameter of 55 mm and issurrounded by a body 42 of the filter unit.

The membrane 41 used here is of cellulose ester and has a pore sizeadapted to retain the microorganisms whose presence it is desired todetect, most often between 0.10 and 100 microns.

The drawer 5 has a body 30, a collar 31 and a grasping lug 32.

In the body 30 there is formed a cylindrical cavity 33 provided toreceive a filter unit 40.

The illuminating means 3 will now be described with the help of FIGS. 3to 5.

These means 3 comprise two separate illuminating members 4.

Each member 4 comprises a mounting 20 of trapezoidal section, a printedcircuit board 21 on which two groups 27 and 28 of diodes 22 and 23 aredisposed, a diffuser 24 covering those diodes and two black screens 26between the edges of the board 21 and those of the diffuser 24.

The mountings 20 are fixed inside the casing 2 on the wall 10 of thatcasing, while the boards 21 are fixed to the mountings 20 on theinclined faces thereof, that are remote from those disposed against thewall 10 such that those boards are inclined towards each other in thedirection of the reception zone 33 for the support 41.

Each mounting 20 is thus provided such that each plate 21 has aninclination A (FIG. 1) of 45° relative to the wall 10 and to thepredetermined location that the membrane 41 occupies, when the unit 40is in the housing 33 of the device (FIG. 1).

The upper edges 25 of the boards 21 are at a distance from each otherequal to 100 mm while those edges 25 are at a distance of 23 mm from thewall 10.

The summits of light emitting diodes 22 and 23 are situated at adistance of 15 mm from the diffusers 24.

These diffusers 24 are here made from a plate of glass on which one ofthe faces is sandblasted (the face which is turned towards the diodes).

On each printed circuit board, and as illustrated in FIG. 5, a firstgroup 27 of diodes is disposed, constituted by sixteen diodes 22 as wellas a second group 28 of diodes constituted by four diodes 23.

The diodes 22 are regularly spaced from each other and disposed in fourequidistant rows of four diodes, such that each diode 22 is situated ata distance of 16 mm from the diodes 22 directly neighboring thereto.This group 27 thus forms an array of diodes making it possible touniformly illuminate the membrane 41 when it is disposed in the casing 2at its predetermined location, the center 29 of that array beingsituated at a height H of 54.75 mm from that predetermined location(FIG. 1).

The diodes 23 are distributed in the four corners of a square interlacedin the center of the array of diodes 22, each diode 23 being situated ata distance of 32 mm from the two diodes 23 neighboring thereto and atthe center of a square at the corners of which are situated four diodes22 (FIG. 5). The center of this array coincides with the center 29 ofthe array constituted by the diodes 22.

The diodes 22 are diodes from the company LUMILED® commercialized underthe reference LXHL-PB01 and of which the emission spectrum 55 isillustrated in FIG. 7, this spectrum of Gaussian distribution formhaving a crest 55′ at the wavelength λ₁ of 470 nm, thus emitting inblue.

The diodes 23 are diodes from the same company commercialized under thereference LXHL-PD01, of which the emission spectrum, not illustrated inthe drawings, is also of Gaussian distribution form and has a crest atthe wavelength of 625 nm thus emitting in red.

These diodes have a solid angle of emission of 140° for a relativeintensity value of 25% and a solid angle of emission of 90° for arelative intensity value of 60%.

The diodes 23 have an emission power substantially four times greaterthan that of the diodes 22 such that they are four times less numerousthan the diodes 22.

The arrays of diodes 22 and 23 are interlaced (that is to say nestedwithin each other) so as to obtain the most homogenous light possible atthe filter unit to be illuminated, whether the light comes from thediodes 22 or from the diodes 23.

Each group of diodes produces a light spectrum centered on apredetermined wavelength so as to be able to excite differentpredetermined types of fluorophore.

The conductive tracks on each board 21 are electrically linked to acommand and control unit for the device (not shown).

The preparation of a sample to analyze will now briefly be described.

Prior to the actual detection step, the operator collects a sample toanalyze (which may contain microorganisms) by filtration through amembrane 41 of a unit 40.

Once the microorganisms have been filtered and retained on the membrane,an optional step of growing the microorganisms in contact with anappropriate growth medium may be included. This growth medium ispreferably a gel medium on which the membrane is deposited afterfiltration. This step, which is optional, enables colonies of each ofthe microorganisms initially filtered to be obtained, which increasesthe number of cells to detect.

The membrane and the microorganisms that it contains are then placed incontact with a composition for rendering the walls of the microorganismspermeable and the fluorophore markers are then incorporated into thepermeable-rendering composition in order to enter the inside of themicroorganisms to detect.

A description will now be given of the implementation of thedetermination of the presence or absence of microorganisms on a sampleso prepared on the basis of the device according to the invention.

In a first phase, the drawer 5 is pulled and the operator places afilter unit 40 (which may possibly be covered by a transparent cover 43to protect the membrane from exterior contamination) in the cavity 33 ofthat drawer. The drawer 5 is then pushed until the collar 31 abuts thewall 13 such that the membrane 41 is disposed in its predeterminedlocation within the casing 2 in order to be illuminated.

Depending on the fluorophore which was used to mark the microorganisms,the operator next selects (for example via a switch not illustrated inthe drawings) the corresponding diodes 22 or 23 to turn on (the diodes22 in the example illustrated) so as to uniformly illuminate the wholeof the surface of the membrane 41 and to excite, at the rightwavelength, the fluorophore that served for the marking.

The diffusers 24 as well as the spatial distribution of the diodes onthe boards 21 make it possible to have a particularly homogenousillumination of the whole surface of the membrane 41, whatever the groupof diodes used.

As the boards 21 are disposed one on each side of the opening 9receiving the filter 6, the light coming from the filter unit 40 whichis emitted in response to the excitation light from the diodes passesthrough that filter as illustrated in FIG. 1 such that it is possible toobserve the light response of the membrane 41 through the filter 6, withthe naked eye or else via a camera.

The light response obtained in the presence of microorganisms using thedifferent spectral diagrams illustrated in FIG. 7 will now be detailed.

In FIG. 7, the spectrum 55 is of Gaussian form and corresponds to thespectrum of the excitation light (here the spectrum produced by thediodes 22) and which has a peak of which the crest 55′ corresponding tothe maximum light intensity value is at the wavelength λ₁ equal to 470nm.

The spectra 53 and 54 respectively correspond to the absorption spectrumand to the emission spectrum of the fluorophore chosen here to mark themicroorganisms present on the membrane.

The fluorophore represented here is 5-6 CFDA(Carboxy-Fluorescein-Di-Acetate).

The absorption spectrum 53 has a crest 53′ at the wavelength λ₂ greaterthan λ₁ and the emission spectrum 54 has a crest 54′ at the wavelengthλ₃ greater than λ₂.

In the example illustrated λ₂ is equal to 492 nm and λ₃ to 517 nm, thedifference between λ₁ and λ₂ here being less than the difference betweenλ₂ and λ₃.

The excitation wavelength λ₁ is deliberately chosen to be less than λ₂such that the crest 55′ of the spectrum 55 is offset relative to thecrest 53′ of the spectrum 53, on the opposite side to the spectrum 54.This offset is chosen in order for the light coming from theilluminating means to have sufficient energy to excite the fluorophorewithout that light causing significant parasitic interference with thatemitted by the fluorophore in response to that excitation light.

The spectrum 56 is that of the filter 6 of the viewing window, itcut-off frequency being chosen (here of the order of 550 nm) to letthrough essentially the light emitted by the fluorophore (spectrum 54)and to stop the light at shorter wavelengths, in particular those comingfrom the diodes 22 after reflection on the unit 40.

This output filter (a colored filter) is weakly selective to allow asufficient quantity of light to return to the eyes of the user giving agenerally bright scene and which is thus comfortable to observe whileensuring a level of contrast which is adapted for observation with thenaked eye.

When the excitation light coming from the illuminating means 3illuminates the membrane 41 of the filter unit 40, each location of thatmembrane having microorganisms marked by the fluorophore is renderedvisible in the form of a bright spot of small size (a few hundreds ofmicrons) directly observable with the naked eye coming out of the filter6.

The values of the angle A and of the height H provide an optimumrendering for the reading of the bright spots on the membrane 41,whether with the naked eye or via a camera.

These values have been determined by applying a black mark and afluorescent yellow mark to a control membrane and by seeking, fordifferent values of that angle A and of that height H, theconfigurations A, H for which the brightness of the fluorescent band ismaximum while minimizing the parasitic brightness on the black mark.

It has thus surprisingly proved that the range [40°-50°] for the angle Agave both a maximum light intensity for the fluorescent mark andpractically an absence of parasite light for the black mark, which thuscorresponds to an optimum in terms of reading comfort.

So as to have a certain safety margin relative to the play in mountingthe device it is thus the average value of 45° which has been chosenhere.

As regards the height value H, this is a function of the size of theobject to illuminate, the different configurations tested have shownthat for a membrane of a diameter of 55 mm and for a value range for theangle A between 40° and 50°, the best results were obtained for a heightrange between 39.75 and 69.75 mm. Similarly here, it is the averagevalue of 54.75 mm which was chosen in this example.

The device is also adapted, by adjusting the height H, to illuminatetypes of sample other that membranes, and generally any support adaptedto contain microorganisms (whether on a surface or in a volume) andwhose presence it is desired to detect by fluorescence.

Another embodiment of this device is represented in FIG. 6.

Generally speaking, the same reference numbers increased by 100 are usedfor similar parts.

The device 101 has the same features as the device 1 apart from itlacking the drawer 5 (the filter unit 40 here being directly placed on atable for example) and for the fact that the viewing window 107 is hereformed from a slide 108 of rectangular form disposed against the wall110 of the casing 102 and from four filters 106 to 106′″, each of thefilters being housed in a corresponding opening of the slide 108.

Each optical filter is a low-pass filter, thus allowing the lowfrequencies to pass (the longest wavelengths) and of which the cut-offfrequency is distinct from the other cut-off frequencies.

The slide 108 is engaged in a guide rail (not illustrated) of thatdevice so as to be able to place any chosen one of the four filters 106to 106′″ in register with the opening 109 of the wall 110.

It is thus possible, depending on the fluorophore chosen and on thediodes which are turned on, to select from the four available filtersthe one which has the most adapted cut-off frequency (to obtain the bestcontrast for example).

In one embodiment not illustrated, the filter 6 is not a low-pass filterbut a band-pass filter of which the passband is centered on thewavelength λ₂.

In still another embodiment not illustrated, the printed circuit boards21 have either a single group of regularly spaced diodes, or more thantwo groups of diodes, each group of diodes being adapted to illuminatethe support 41 with different wavelengths, the arrays of each group ofdiodes having regular shapes, such as a square, diamond, triangle, etc.

In still another embodiment not illustrated, the diodes are replaced byany other type of localized and unitary light source such aschemiluminescent light spots and/or the printed circuit boards arereplaced by any other type of base able to support light sources such asglass or ceramic plates.

Lastly, it will be noted that such an illuminating system may also beproduced in different versions for many other applications such asanalysis by microscope, scanning of biochips, reading of plates byfluorescence, cytometry, transilluminators, PCR real-time reading, thegeometrical configuration of the device for each of these applicationsthen being in a version specific to the application in question. Theframe of the device to which the bases 21 are fixed is not necessarily acasing like the casing 2 illustrated; it may for example be a hoopsurrounding the optics of a microscope.

The present invention is not limited to the embodiments described andrepresented, but encompasses any variant form thereof.

The invention claimed is:
 1. An assembly comprising a device for themicrobiological analysis of a support having a surface containingmicroorganisms marked by a fluorophore agent; said assembly comprising,as said support, a microporous membrane having a diameter of 55 mm orbelonging to a filter unit having a body surrounding said membrane, saidfilter unit having a transparent cover to protect said microporousmembrane, and an illuminator adapted to illuminate said support todetect, by fluorescence, the presence of said marked microorganisms aswell as a window for viewing light emitted by said support in responseto excitation light produced by said illuminator; wherein saidilluminator comprises two bases on each of which is mounted at least onegroup of light sources, with said sources of that group being regularlyspaced from each other to form a first array for illuminating saidsupport, said bases being fixed to a frame of said device, one on eachside of an opening formed in said frame to allow the light emitted bysaid support to pass through said viewing window, with said bases beinginclined towards each other in the direction of a predetermined locationfor reception of said support in said device, wherein said illuminatorcomprises, for each base, a diffuser for the light emitted by saidsources, there being only said diffuser between said group of lightsources and said support, and wherein on each base there is also mountedin addition to said group of light sources, designated a first group,another group of light sources, designated a second group, with saidsources of said second group also being regularly spaced from each otherto form a second array for illuminating said support, the first andsecond arrays being interlaced within each other, and wherein saidilluminator is configured in said device to illuminate the whole surfaceof said support, enabling observation of the light response of saidsupport with a naked eye, and wherein light emitted from said first andsecond groups of light sources has an excitation spectrum crest of λ₁,said fluorophore agent has an absorption spectrum crest at λ₂ and anemission spectrum crest at λ₃, where λ₁<λ₂<λ₃ and (λ₂−λ₁)<(λ₃−λ₂), andsaid viewing window comprises a selective filter that blockssubstantially all light from passing through the filter up to a firstwavelength, allows substantially all light to pass through the filter atand above a second wavelength, thereby defining a transition regionbetween said first and second wavelengths, and wherein λ₂ and λ₃ arewithin said transition region and λ₁ is not.
 2. A device according toclaim 1, wherein said light sources each have a same light spectrumcentered on a predetermined wavelength.
 3. A device according to claim2, wherein said light sources are light emitting diodes.
 4. A deviceaccording to claim 1, wherein the height (H) between the center of eacharray and said predetermined location for reception of said support insaid device is a function of said support.
 5. A device according toclaim 1, wherein for each base, the centers of said first and secondarrays coincide.
 6. A device according to claim 1, wherein each diffuseris a plate of glass on which one of the faces is sandblasted.
 7. Adevice according to claim 1, wherein said viewing window comprises afilter adapted allow the longest wavelengths to pass.
 8. A deviceaccording to claim 7, wherein said viewing window comprises a pluralityof filters.
 9. A device according to claim 8, wherein said filtersbelong to a slide slidingly mounted on said frame.
 10. A deviceaccording to claim 1, wherein said illuminator is adapted to illuminatethe whole of said predetermined location for reception of said support.11. A device according to claim 10, wherein said support comprises amicroporous membrane.
 12. A device according to claim 1, wherein eachsaid base is inclined by an angle (A) between 40° and 50° relative tosaid predetermined location for reception of said support, wherein thecenter of each said array is situated at a height (H) between 39.75 mmand 69.75 mm from said support, each said base has an upper edge, andwherein the upper edges of said bases are spaced a distance of 100 mmfrom each other.